We have investigated whether Plx1, a kinase recently shown to phosphorylate cdc25c in vitro, is required for activation of cdc25c at the G2/M-phase transition of the cell cycle in Xenopus. Using immunodepletion or the mere addition of an antibody against the C terminus of Plx1, which suppressed its activation (not its activity) at G2/M, we show that Plx1 activity is required for activation of cyclin B-cdc2 kinase in both interphase egg extracts receiving recombinant cyclin B, and cycling extracts that spontaneously oscillate between interphase and mitosis. Furthermore, a positive feedback loop allows cyclin B-cdc2 kinase to activate Plx1 at the G2/M-phase transition. In contrast, activation of cyclin A-cdc2 kinase does not require Plx1 activity, and cyclin A-cdc2 kinase fails to activate Plx1 and its consequence, cdc25c activation in cycling extracts.

REFERENCES

Abrieu
A.
,
Lorca
T.
,
Labbe
J. C.
,
Morin
N.
,
Keyse
S.
,
Doree
M.
(
1996
).
MAP kinase does not inactivate, but rather prevents the cyclin degradation pathway from being turned on in Xenopus egg extracts.
J. Cell Sci
109
,
239
246
Abrieu
A.
,
Fisher
D.
,
Simon
M. N.
,
Doree
M.
,
Picard
A.
(
1997
).
MAP kinase inactivation is required for the G2to M-phase transition in the first mitotic cell cycle.
EMBO J
16
,
6407
6413
Brown
G. C.
,
Hoek
J. B.
,
Kholodenko
B. N.
(
1997
).
Why do protein kinase cascades have more than one level.
Trends Biochem. Sci
22
,
288
–.
Clarke
P. R.
,
Leiss
D.
,
Pagano
M.
,
Karsenti
E.
(
1992
).
Cyclin A-and cyclin B-dependent protein kinases are regulated by different mechanisms in Xenopus egg extracts.
EMBO J
11
,
1751
1761
Devault
A.
,
Cavadore
J. C.
,
Fesquet
D.
,
Labbe
J. C.
,
Lorca
T.
,
Picard
A.
,
Strausfeld
U.
,
Doree
M.
(
1991
).
Concerted roles of cyclin A, cdc25 mitotic inducer, and type 2A phosphatase in activating the cyclin B-cdc2 protein kinase at the G2/M phase transition.
Cold Spring Harbor Symp. Quant. Biol
56
,
503
513
Devault
A.
,
Fesquet
D.
,
Cavadore
J. C.
,
Garrigues
A. M.
,
Labbe
J. C.
,
Lorca
T.
,
Picard
A.
,
Philippe
M.
,
Doree
M.
(
1992
).
Cyclin A potentiates maturation-promoting factor activation in the early Xenopus embryo via inhibition of the tyrosine kinase that phosphorylates cdc2.
J. Cell Biol
118
,
1109
1120
Doree
M.
(
1990
).
Control of M-phase by maturation-promoting factor.
Curr. Opin. Cell Biol
2
,
269
273
Dunphy
W. G.
,
Kumagai
A.
(
1991
).
The cdc25 protein contains an intrinsic phosphatase activity.
Cell
67
,
189
196
Fenton
B.
,
Glover
D. M.
(
1993
).
A conserved mitotic kinase active at late anaphase-telophase in syncytical Drosophila embryos.
Nature
363
,
637
640
Ferrell
J. E.
Jr.
(
1996
).
Tripping the switch fantastic: how a protein kinase cascade can convert graded inputs into switch-like outputs.
Trends Biochem. Sci
21
,
460
466
Ferrell
J. E.
Jr.
(
1997
).
How responses get more switch-like as you move down a protein kinase cascade.
Trends Biochem. Sci
22
,
288
289
Gautier
J.
,
Solomon
M. J.
,
Booher
R. N.
,
Bazan
J. F.
,
Kirschner
M. W.
(
1991
).
Cdc25 is a specific tyrosine phosphatase that directly activates p34cdc2.
Cell
67
,
197
211
Glover
D. M.
,
Ohkura
H.
,
Tavares
A.
(
1996
).
Polo kinase: the choreographer of the mitotic stage?.
J. Cell Biol
135
,
1681
1684
Golsteyn
R. M.
,
Mundt
K. E.
,
Fry
A. M.
,
Nigg
E. A.
(
1995
).
Cell cycle regulation of the activity and subcellular localization of PLK1, a human protein kinase implicated in mitotic spindle function.
J. Cell Biol
129
,
1617
1628
Hamanaka
R.
,
Smith
M. R.
,
O'Connor
P. M.
,
Maloid
S.
,
Mihalic
K.
,
Spivak
J. L.
,
Longo
D. L.
,
Ferris
D. K.
(
1995
).
Polo-like kinase is a cell cycle-regulated kinase activated during mitosis.
J. Biol. Chem
270
,
21086
21091
Hoffmann
I.
,
Clarke
P. R.
,
Marcote
M. J.
,
Karsenti
E.
,
Draetta
G.
(
1993
).
Phosphorylation and activation of human cdc25-C by cdc2—cyclin B and its involvement in the self-amplification of MPF at mitosis.
EMBO J
12
,
53
63
Huang
C. Y.
,
Ferrell
J. E.
Jr.
(
1996
).
Ultrasensitivity in the mitogen-activated protein kinase cascade.
Proc. Nat. Acad. Sci. USA
93
,
10078
10083
Izumi
T.
,
Maller
J. L.
(
1993
).
Elimination of cdc2 phosphorylation sites in the cdc25 phosphatase blocks initiation of M-phase.
Mol. Biol. Cell
4
,
1337
1350
Jessus
C.
,
Beach
D.
(
1992
).
Oscillation of MPF is accompanied byperiodic association between cdc25 and cdc2-cyclin B.
Cell
68
,
323
332
Kitada
K.
,
Johnson
A. L.
,
Johnson
L. H.
,
Sugino
A.
(
1993
).
Amulticopy suppressor gene of the Saccharomyces G1cell cycle mutant dbf4 encodes a protein kinase and is identified as CDC5.
Mol. Cell. Biol
13
,
4445
4457
Knoblich
J. A.
,
Lehner
C. F.
(
1993
).
Synergistic actin of Drosophila cyclins A and B during the G2-M transition.
EMBO J
12
,
65
74
Kumagai
A.
,
Dunphy
W. G.
(
1992
).
Oscillation of MPF is accompanied by periodical association between cdc25 and cdc2-cyclin B.
Cell
68
,
323
332
Kumagai
A.
,
Dunphy
W. G.
(
1996
).
Purification and molecular cloning of Plx1, a cdc25 regulatory kinase from Xenopus egg extracts.
Science
273
,
1377
1380
Labbe
J. C.
,
Cavadore
J. C.
,
Doree
M.
(
1991
).
M-phase specific cdc2 kinase: preparation from starfish and properties.
Meth. Enzymol
201
,
291
301
Lee
K. S.
,
Erikson
R. L.
(
1997
).
Plk is a functional homolog of Saccharomyces cerevisiae cdc5, and elevated Plk activity induces multiple septation structures.
Mol. Cell Biol
17
,
3408
3417
Masui
Y.
,
Markert
C. L.
(
1971
).
Cytoplasmic control of nuclear behavior during meiotic maturation of frog oocytes.
J. Exp. Zool
177
,
129
145
Morin
N.
,
Abrieu
A.
,
Lorca
T.
,
Martin
F.
,
Doree
M.
(
1994
).
The proteolysis-dependent metaphase to anaphase transition: calcium/ calmodulin-dependent protein kinase II mediates onset of anaphase in extracts prepared from unfertilized Xenopus eggs.
EMBO J
13
,
4343
4352
Mueller
P. R.
,
Coleman
T. R.
,
Dunphy
W. G.
(
1995
).
Cell cycle regulation of a Xenopus wee1-like kinase.
Mol. Biol. Cell
6
,
119
134
Mueller
P. R.
,
Coleman
T. R.
,
Dunphy
W. G.
(
1995
).
MYT 1: a membrane-associated inhibitory kinase that phosphorylates cdc2 on both threonine 14 and tyrosine 15.
Science
270
,
86
90
Murray
A. W.
,
Kirschner
M. W.
(
1989
).
Cyclin synthesis drives the early embryonic cell cycle.
Nature
339
,
275
280
Nurse
P.
(
1990
).
Universal control mechanism regulating onset of M-phase.
Nature
344
,
503
508
Ohkura
H.
,
Haggan
I.
,
Glover
D. M.
(
1995
).
The conserved Schizosaccharomyces pombe kinase plo1, required to form a bipolar spindle, the actin ring, and septum, can drive septum formation in G1and G2cells.
Genes Dev
9
,
1059
1073
Russell
P.
,
Nurse
P.
(
1986
).
Cdc25+functions as an inducer in the mitotic control of fission yeast.
Cell
45
,
145
153
Solomon
M. J.
,
Glotzer
M.
,
Lee
T. H.
,
Philippe
M.
,
Kirschner
M. W.
(
1990
).
Cyclin activation of p34cdc2.
Cell
63
,
1013
1024
Sunkel
C. E.
,
Glover
D. M.
(
1988
).
Polo, a mitotic mutant of Drosophila displaying abnormal spindle poles.
J. Cell Sci
89
,
25
38
Strausfeld
U.
,
Labbe
J. C.
,
Fesquet
D.
,
Cavadore
J. C.
,
Picard
A.
,
Sadhu
K.
,
Russell
P.
,
Doree
M.
(
1991
).
Dephosphorylation and activation of a p34cdc2/cyclin B complex in vitro by human cdc25 protein.
Nature
351
,
242
245
Strausfeld
U.
,
Fernandez
A.
,
Capony
J. P.
,
Girard
F.
,
Lautredou
N.
,
Derancourt
J.
,
Labbe
J. C.
,
Lamb
N. J.
(
1994
).
Activation of p34cdc2protein kinase by microinjection of human cdc25 into mammalian cells. Requirement for prior phosphorylation of cdc25c by p34cdc2on sites phosphorylated at mitosis.
J. Biol. Chem
269
,
5989
6000
Tavares
A. A.
,
Glover
D. M.
,
Sunkel
C. E.
(
1996
).
The conserved mitotic kinase Polo is regulated by phosphorylation and has preferred microtubule-associated substrates in Drosophila embryo extracts.
EMBO J
15
,
4873
4883
Walter
S. A.
,
Guadagno
T. M.
,
Ferrell
J. E.
(
1997
).
Induction of a G2-phase arrest in Xenopus egg extracts by activation of p42 mitogen-activated protein kinase.
Mol. Biol. Cell
8
,
2157
2169
Westendorf
J. M.
,
Swenson
K. I.
,
Ruderman
J. V.
(
1989
).
The role of cyclin B in meiosis I.
J. Cell Biol
108
,
1431
1444
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